Preparation of alpha-1,6-glucosidase

ABSTRACT

A-1,6-GLUCOSIDASES WHICH ARE CAPABLE OF SELECTIVLEY HYDROLYZING THE ALPHA-1,6-GLUCOSIDE BONDES OF STARCH AND WHICH HAVE HIGH HEAT RESISTANCE AND ACTIVITY ARE PRODUCED FROM STRAINS OF BACTERIA BELONGING TO THE GENUS ARGOBACTERIUM. AZOTOBACTER, BACILLUS, MYCOBACTEIUM, NOCARDIA, LEUCONOSTOC, MICROCOCCUS, MYCOBACTERIUM, NORCADIA, PEDIOCOCCUS, SARCINA, SERRATIA, STPHYLOCOCCUS AND STREPTOCOCCUS.

Activity Activity E 620 ljl E 620 alu KANAME/ SIJGHMOTO ETAL PREPARATION' OF-l ,'6-GLUCOSIDASE original Filed liarnn 2 5, 1969 locnrdin *g licrococus Lactobacillus /v /n -lb-I- /v/l 3o 4o 50 INVENTORS 'Wm- Temperature C) 7211411114, W

h JMW ATfoRNEYs' United States Patent t U.S. Cl. 195-66 R 4 Claims ABSTRACT OF THE DISCLOSURE a-l,6glucosidases which are capable of selectively hydrolyzing the alpha-1,6-glucoside bonds of starch and which have high heat resistance and activity are produced from strains of bacteria belonging to the genus Agrobacterium, Azotobacter, Bacillus, Erwinia, Lactobacillus, Leuconostoc, Micrococcus, Mycobacterium, Nocardia, Pediococcus, Sarcina, Serratia, Staphylococcus and Streptococcus.

This is a continuation of application Ser. No. 810,293, tiled Mar. 25, 1969, now abandoned.

a1,6glucoside bonds of starch have long been known. In recent years they have aroused interest in connection with identification of starch structures, and have become a subject of extensive study. It is known that these enzymes have somewhat different activities and are classiable for that matter into three types, as in Table l.

TABLE 1 [Types of PLS-glucosidase and their substrate speciticity] Enzyme source Yeasts Bacteria Rice, broad beans,

potato and barley. Name of enzyme Isoamylases.. Pullulanases- R-enzymes. Speecity to substrates:

starches -limit dextrins Glyomrens Dextrans Pnllnlans Each of the three types of different activities as summarized in the table exhibits specificity to various types of a-l,6glucoside bonds. For example, isoamylases of yeasts will not act upon the i6-glucoside bonds of pullulans, pullulanases of bacteria will be mactive against the 1,6-glucoside bonds of dextrans, and R-enzymes of plant origins will not act upon the 1,6-glucoside bonds of glycogens but upon those of starches. Thus,

they differ in the behaviors of enzymatic activities, though -much is to be clarified yet.

The present inventors investigated these known enzymes from the industrial viewpoint because they could be of important significance for utilization on starches and similar decomposition products such as glycogens, dextrans,and pullulans. As a result, it was found that the isoamylases'and R-enzymes obtained from yeasts and plants, respectively, have very small activities and very low optimum temperatures of to 40 C., while the pullulanases of bacteria have relatively high activit1es and are most activeat a slightly higher temperature of 45 AC. Therefore, with the view to finding enzymes of different substrate speciiicity for application demanding greater specicity, or enzymes of improved heat resistance and activity which are both weak points of the existing enzymes, the present inventors searched for a-l,6glu cosidase-productive bacteria out of 169 strains of bacterial type cultures.

3,827,940 Patented Aug. 6, 1974 ice The clue used in the search was the phenomenon that when a strain is productive of an enzyme having an al,6 glucosidase activity, the starch and iodine-potassium iodide solution added will turn blue. This led to the finding that the above enzymes could be produced by 40 strains of 16 genera, namely, Bacillus, Lactobacillus, Nocardia, Agrobacterum, Azotobacter, Leuconostoc, Pediococcus, Mycobacterium, Pseudomonas, Sarcina, Staphylococcus, Serratia, Aerobacter, Erwinia, Streptococcus, and Micrococcus. Of these bacteria, those of the genus Aerobacter have been reported by many students and the use of bacteria of the genus Pseudomonas is taught by copending Patent Application Ser. No. 733,326, now U.S. Pat. No. 3,560,345. Therefore, excepting those of the two genera above mentioned, all strains of the 14 genera on hand were cultured on culture media for aerobic and lactic acid bacteria, of the compositions to be described later, each in an amount of 100 ml. and kept at 30 C. The aerobic strains were cultured by the rotary Vibration method and the lactic acid strains by the stationary method. The enzymatic activity was determined in the following manner using a supernatant fluid obtained by centrifuging each cultured uid produced and also a supernatant fluid obtained by causing autolysis of the precipitated fungus bodies in a butler solution and then centrifuging the solution. Each composition consisting of 5.0 ml. of 1% liquefied glutinous rice starch, 1.0 ml. of 0.5M acetic acid butler solution, and 1.0 m1. of an enzyme solution was reacted at 40 C. for 30 minutes. From the reactant solution, 1.0 ml. portions were sampled after the reaction periods of zero hour and 30 minutes. Each sample portion was poured into a mixture of 30 ml. of 0.01N sulfuric acid and 0.01N iodine-potassium iodide solution. The mixture turned purple, and the extinction coefficient at 620 mp. was determined l5 minutes later. The increment of the extinction coeicient of the reactant solution during a period of 30 minutes from the outset of the reaction was regarded as the enzymatic activity.

In these conditions increase of 0.01E, coetlicient corresponds to l unit of activity.

Of the strains examined for the activity in the manner described, typical strains of the genera which produce more enzymes outside their fungus bodies than inside may be mentioned, one for each genus, as Azotobacter indicas, Bacillus cereus, Erwnia arodeae, and Nocaralar corallina. The strains which produce more enzymes inside the fungus bodies represent ten genera, namely, Agrobacterum tumefacz'ens, Mycobacterium lyso'dektcus, Micrococcus lysodeikticus, Sarcna luta, Serrata plymuthz'ca, Staphylococcus surus, Lactobacillus plantarum, Leuconostoc ctrovorum, Pedococcus acdlactz', and Streptococcus faecalz's.

Particularly active of the strains chosen above were those of the genera Lactobacillus, Nocardia and Micrococcus. They were cultured in jars and purified for the investigation of their enzymatic behaviors and substrate specicty. The culture media used were of the following two compositions:

CULTURE MEDIA Percent For aerobic For lactic bacteria acid bacteria Peptone l 5 Yeast extract 0. 5 0. 1 K2HPO4...- 0.1 0.1 NaCl 0.05 0. 05 MgSO4-7H2O 0.05 0. O5 FeSO4-7H2O. 0.001 0.001 MnSO4-4H2O 0. 0002 Liqueed starch 1. 4 0. 7 Maltese 0. 5

3 4 diluted l-fold, and the pH was determined with the use reference to the accompanying drawings; in which, graphof glass electrodes. ically representing the conditions of enzyme solutions to As for the aerobic strains of the three genera above be used in the practice of the present invention: FIG. l mentioned, they were cultured in jars under aeration, and is a curve showing the optimum pH values; and FIG. 2

the strains of the genera Nocardia and Lactobacillus were cultured at 30 C. for 3 days and the Micrococcus strains D were cultured at 30 C. for 1.5 days. The former two EXAMPLE I were purified through autolysis of the cultured liuids and Production of 0L 1 @glucosidases by bacteria salting out with ammonium sulfate and dialysis of the is a curve showing the optimum temperatures.

supernatant iiuid repeated twice each. The cultured fluid UZIlg tht- OdiHe-Safch reaction, a quest WHS madc of each Micrococcus strain was treated after the complefor Strains Whlchcould del/@10P CPOY 011 that FSL AS a tion of the Culture by collecting the bacteria, washing it result, those strains were found in thebacteria of the with pure Water, causing autolysis for 43 hours with the genera Aerobacter, Bacillus, Lactobacillus, Nocardia, addition of an S.D.S. buffer solution, centrifuging, and Aglobaefium, Azoobactef, LCUCODOSOC, Lactobacillus, subjecting the supernatant uid to salting out with am- Pediococcus, Strepococcus, Micrococcus, Microbacterimonium sulfate, and thereby extracting a soluble enzyme.

ENZYMATIC BEHAVIOR AND SUBST RATE SPECIFICITY Enzyme Isoamylases of Pullulanases of Enzymes of R-enzymes of Enzymes of Enzymes of Enzymes of Enzymatic property yeasts Aerobaeter Pseudomonas plant; origins Nocarda Lactobacillus Microeoecus Optimum pil--. 6-6. 5.5-6 5-6 5.5-6 Optimum temp C C liti-4:5" Over 50 C 45 C. pH stability pH 6-9; pH 5-8; I pH 6.5; pH 5-8;

rapidly derapidly deaetivd. at rapidly derapidly derapidly deactivd. at actvd. at under pH 5. activd. at activd. at actvd. at under pH 5. under pH 5. under pH 5. iider pH under pH 5. Heat stability Rapidly de- Rapidly de- Rapidly de- Rapidly de- Rapidly de- Rapidly deactvd. at actvd. at actvd. al; actvd. at aotvd. at activ at over C. over 45 C. over 45 C. over 45 C. over 57 C. ver t0-45 Substrate specificity: n

Potatostarch. -l- -ll- -l Gliitiucus rice starch.- -l- -i- -l- -l- -l- The enzyme solutions were tested for their enzymatic properties and gave results as tabled. For reference, comparative results of isoamylases of yeasts, pullulanases of the genus Aerobacter, and enzymes of the genus Pseudomonas are also shown. Considering these results, it is apparent that the Lactobacillus enzymes have higher 40 optimum temperatures and heat stability, i.e., with opities.

um, Pseudomonae, Sarcina, Serratia, and Staphylococcus.

Therefore, the strains on hand which belong to these genera were cultured in flasks and were tested for activ- One hundred milliliters of either culture medium of the following composition was inoculated with a small platinum spoonful of each test strain, and cultivation was timum temperatures of over 50 C. in contrast to the carried 0pt for 4 daYS- The imperia-ture Wa 5 kept at. 30 range of 40 to 45 o C. for the other enzymes and hence Aerobic bacteria were cultured with rotation and vibraare more adapted for commercial applications than the non Whlle lactic amd bacrerla Were cultured Still rest. As for the optimum pH and the pH stability, the enzymes of the genus Nocardia display optimum pH CULTURE MEDIA values in the range of 6 to 7, or slightly higher than those Percent of the Lactobacillus and Micrococcus enzymes which For aerobic For lactic are in the neighborhood of 5.5. In the aspect of pH stabacteria acid bacteria bility, all of the enzymes rapidly lose ltheir activities at Femm 1.o 1 0 -pH 5 or downwards, excepting the Lactobacillus enzymes Yeast extract 0.5 0,5 KgHPoi 0.1 0.1 which remain active at relatively low pH values. Nam 0.05 0 05 Substrate specificity was evaluated by subjecting a sub- MgSOiJHzO 0.05 0.05 strate to the action of -ainylase as Well as of the particufgsf: o of lar a-1,\6-glucosidase and then determining the decompo- 55 Maitose-- Y 0 5 sition rate. Substrates used for this purpose were potato starch, glutinuous rice starch, -limit dextrin, glycogen, dextran, and pullulan. The decomposition rate of pul- The result? 0f Culture Were aS ablefr below, 111 WhlCl-l ulan was Compared in terms of the produc/[ign rate of only the strains of relatively great activities are shown: malt-triose and the decomposition rates of others were compared in terms of the production rates of maltose. The .Activity results, as tabled herein, indicated that the three types of Outside Inside enzymes are invariably active against the 1,6-bonds of fungus fungus Tom the starch system, they are all inactive to dextrans, and Test strain body body activity that the enzymes of the genus Micrococcus alone exhibit Agmbmm-ummmfumm m0 mm 9.3 0 2.3 23 no activity against glycogens. In this sense, the Micro- Agvgvder indicas IFO 3744 ATCC 7 l 1 1 o 1 1 cocus enzymes may be likened i0 R-enzymes 0f plant Bama.;'iddfSIIIIZIIIIII s 517 4.o 921 origins so far as ithe type of activity against al,6glyco gggn'gfgargTcc 8247-- g- -g Sig-g sides is concerned. The other enzymes of the genera Micromania lyaodeikttcus "I'15"` Lactobacillus and Nocardia are considered akin to the MgbCage-Egri::n:: g ag gig pullulanases of the genus Aerobacter. The invention will Numara corallina iFo asas Arco now be more fully described in conjunction with exam- Saggig'fgj "I: g1g 248 jg zg' ples thm-eci .sarczjna luren info 3232.-.-. 9.2 o 4. 7 4.7 Saremo vcrabzlza IFO 3067. 8. 8 0 4. 6 4. 6 The present invention will now be more fully described semina indica 1ro 3759 3.3 a 2:2 2.3:

TABLE-Continued Activity Outside inside fungus fungus Total Test strain ody body activity Staphylococcus aureus:

IFO 3332 9. 2 0 4. 5 4. 5 IFO 3761 ATCC 4012 8. 7 0 3.0 3. 0 IFO 3340 9. 2 0 5.0 5.0 Lactobacillus brebis:

IFO 3345 ATCC 8287 4. 7 2. 1 1. 5 3.6 IFO 3960 4.8 0. 3 2. 7 3.0 Lactobacillus bulgaricux 3. 9 1. 3 3. 6 4. 9 Lactobacillus fermentum ATCC 8289- 6. 5 0 2.0 2. 0 Lactobacillus plantarurn ATCC 8008.. 3.8 3. 5 3. 9 7. 4 Leuconostoc ctrovorum ATCC 8081..-- 4. 0.8 2. 0 2. 8 Leuctmostoc mesenterodes IFO 3426 ATCC 9135 4. 4 0.3 3.1 3. 4 Pediococcus actdlactz'ci IFO 3884 4. 0 0. 1 2. 3 2. 4 Streptococcus faecium:

IFO 3128 NRRLB-446 4.1 1. 8 2. 4 IF() 3181 ATCC 8043 4. 1 1. 8 1. 9 3 7 EXAMPLE II Production of enzymes from Lactobacillus plantarum and Nocarda corallina and study of their properties The same culture media as used in Example I were employed, and cultivation was carried out for each liter at 30 C. for 3 days with shaking. The results were as follows:

The test enzyme solution of the Lactobacillus bacteria was prepared by saturating the supernatant uid of the cultured solution with ammonium sulfate to a 0.8 saturated solution, centrifugally collecting the resulting precipitate, dissolving the collected precipitate, and dialyzing the same with water for one day.

The cultured fluid of the Nocardia bacteria was similarly treated. The supernatant uid was saturated with ammonium sulfate to form a 0.8 saturated solution, and the precipitate was centrifuged, dissolved in water, devoided of insoluble matter dialyzed and was then saturated to 0.8 with ammonium sulfate, salted out, and dialyzed. The enzyme solution thus obtained was tested. When the optimum pH values were determined, as shown in FIG. l, the Nocardia enzyme solutions displayed pH values between 6 and 7, while the Lactobacillus solutions gave values between and 6.5. Whereas the optimum temperatures for the Nocardia solutions ranged from 42 to 46 C., as shown in FIG. 2, those for the Lactobacillus solutions were upwards of 55 C., thus indicating the greater heat resistance of these enzymes than those of the other enzymes. By contrast, the Nocardia enzymes rapidly lost their activities at 50 C.

For the determination of substrate specificity of the enzymes, substrates prepared in the applicants laboratory and whose purity was conrmed by paper chromatography, namely, potato starch, glutinous rice starch, limit dextrin, dextran, glycogen, and pullulan, were used. These substrates were subjected to simultaneous actions of each test enzyme and -amylase, and the results were expressed in terms of the production rates of maltose of malt-triose, as in Table 2. As can be seen from the table, the Nocardia enzymes do not decompose dextran and the Lactobacillus enzymes are also incapable of decomposing the same.

TABLE 2 [Activities against substrates (-amylo1ysis)] Enzyme ptt-amylase -amylase d and The values in the table represent the percentage of reduced sugars (maltose and malt-triose) in the total sugar.

Total sugar was estimated by the anthrone method and the reducing sugars by the Somogyi-Nelson method. Test specimens of -limit dextrin, -amylase, maltose, pullulan, and malt-triose used were prepared at the applicants laboratory and were of the purity confirmed by paper chromatography.

EXAMPLE III Production of enzyme from M crococcus lysodektcus and study of the enzymatic behaviors An example of culture of 20 l. in a jar is cited here. As the culture medium, a composition consisting of 1% maltose, 0.5% peptone, 0.25% yeast extract, 0.2% urea, 0.2% meat extract, 0.1% K2HPO4, 0.05% KCl, and 0.05 MgSO4-7H2O was inoculated with 2% bacteria at 30 C., and the culture was carried out with aeration and stirring at 200 r.p.m. The initial pH was 7.0. Three days later the culture was terminated with pH 8.2. While there was still a large proportion of bacterial enzyme, the bacteria was centrifugally collected and washed once with pure water. A 0.1% S.D.S. solution containing a buier solution of pH 7.0 was suspended in one-tenth by volume of the culture fluid, and autolysis was eiiected in a rotary shaker at 30 C. for 48 hours. Following centrifugal separation, the supernatant iiuid was salted out with 0.8 saturation with ammonium sulfate. From the resultant precipitate, the enzyme was extracted with a suitable amount of water and then the extracted enzyme solution was dialyzed. On completion of the culture, the endo-enzyme activity was 39.2 units/ml. and the exo-enzyme activity was 12 units/ml. With the enzyme solution thus purified the behaviors were investigated. The optimum pH and the optimum temperature were found to be about pH 5.5 and about 42 C., respectively, as shown in FIG. 1 and FIG. 2. At temperatures above 50 C., the enzyme sharply lost is activity. Also, at pH values of less than 5, it was rapidly deactivated.

As for substrate specificity, the enzyme was tested in the same manner as described in Example II. As shown in Table 3, the results indicated that it was not active upon dextran and glycogen. To confirm the behavior, the enzyme was additionally tested for the activity against glycogen. The results are given in Table 4.

TAB LE 3 [Substrate specllicity] The speclc activities of the enzymes of the genera-Mierococeus, Aerobaeter and Pseudomonas against various substrates were compared, the numerical values being given in terms of the decomposition ratos in 2. A process in accordance with claim 1 wherein said strain is Lactobacillus brevz's IFO 3345 or IPO 3960. Lactobacillus fermentum ATCC 8289 or Lactobacillus plantarum ATCC 8008.

reent. pe 5 3. A process for producing a-L-glucosidase capable of Substrate selectively hydrolyzing the :x16-glucoside bonds of starch Gmb dfilif and which has an optimum pH of 6-7, comprising:

nous (waxy Potato rica com De Glyn PHL lo moculatmg a'lngh optimum pH a 1,6 glucosldnse pro Enzyme starch starch starch) tran gen lulan ducmg strain belonging to the genus Nocardla mto a -Amylase 57 8 0.7 37 o culture medium containing a source of nitrogen and -Amylase and the enzyme- 9 7s s4 1.5 35 115 a Source 0f Carbon, 'fgfeafd culturing the strain until a yield of said high optimum 'h'm" 84 86 84 0 55 99 15 pH a-1,6glucosidase is obtained; and

Pseudomonas enzyme 91 83 59 a 4 S1 o recovering said hlgh optimum pH a 1,6 glucosidase.

No'rn.-M=abbrevlation oi a Mierococcus enzyme.

TABLE 4 [Results oi additional test on the enzymatic activities against glycogen] Enzyme -Amylese -Amylase and the and aeroenzyme bacter- -Arnylaso MI enzyme Remarks 1- 36. 8 35. 4 54. 7 Acts simultaneously with -amylase (MI enzyme 100 p/g. substrate).

2 3l. 7 Acts simultaneously with -amylase (MI enzyme 100 ulg. Substrate).

31. 7 34. Acts simultaneously with tl-amylase (MI enzyme` 250 alg. substrate).

31.7 Acts simultaneously with -amylase, and deactivated 24 hours later (MI enzyme 250 ulg.' substrate), and then acts simultaneously with -amylase under the same conditions before the deactivation.

3 33. 3 33. 4 43. 3 Acts simultaneously with -amylase (MI enzyme 100 ulg. substrate).

What is claimed is:

1. A process for producing L6-glucosidase capable of 40 4. A process in accordance with claim 3 wherein said strain is Norcardia corallina, IFC-3338.

References Cited Walker et. al., Metabolism of the Reserve Polysaccharide of Streptococcus mitis. Biochem. I., vol. 105, 1967 (pp. 93?942) QP5o1.B47.

Veda et al., Production of Isoamylase by Escherichia intermedia. Applied microbiology, vol. 15, No. 3, May 1967 (pp. 492-496) QRIA6.

Dixon, et al., Enzymes, 2nd ed., Academic Press Inc., N.Y., 1964 (pp. 33, 34, 39, 46, 742 and 743) QP601.

DAVID M. NAFF, Primary Examiner U.S. Cl. XR. 195--62 

